DESC:FIELD OF INVENTION
The present invention relates to an Ultra Performance Liquid Chromatography (UPLC) based method for separation and analysis of glycan species.
BACKGROUND OF INVENTION
Glycans are important biomolecules made up of several monosaccharaides linked together glycosidically. In nature they exist either independently or conjugated to other biomolecules such as lipids or proteins as side chains. Based on the branching pattern these could be a simple single chain mono-antennary structure or, highly complexed and branched di- tri- and multi- antennary structures. Predominantly they play a vital role in structural organization of cells and, also as a recognition ligand for several cell-cell, cell-microbe, and antibody microbe interactions. They facilitate several cellular events such as intercellular as well as intracellular signaling, cell development, immune recognition etc.
Glycans are responsible for biological activity of many biomolecules such as antibodies, hormones, enzymes etc. Several immunoglobulins owe their antibody dependent cytotoxicity (ADCC) and cell dependent cytotoxicity CDC to their inherent unique glycan profile. Glycans are also responsible for the biological activity and therapeutic efficacy of many proteins based and small molecular drugs such as antibiotics, cardiotonics, anti-cancers, antibodies, erythropoietin and many more.
Any alteration or removal of glycans have severely impacted the efficacy of many recombinant therapeutic proteins. Glycan constituents of a therapeutic biomolecule impact the performance of the same. Thus assessing the glycan content of biomolecules is important for diagnosis of disease as well as characterization of therapeutic products.
An industrial (“large scale, or industrial scale”) method for glycan profiling of a product is a liberated glycomic procedure in which the glycans are released from the non-glycan portion of the molecule by digesting it with enzymes such as peptide-N-glycanase. The individual glycan species are further separated chromatographically by using HPLC and are characterized by mass spectrometry, NMR or calibrated using retention time of suitable glycan standards.
The separation of glycans is routinely done chromatographically using HPLC (High Performance Liquid Chromatography) based techniques that usually take considerable amount of time leading to an increased turnaround time for the analysis of each sample and reduced number of determinations. Also, there is a need to provide an optimized process for effective separation of glycans to characterize the glycan profile of said samples. In order to overcome the above limitations the present invention describes a rapid and sensitive UPLC (Ultra Performance Liquid chromatrogphy) based method for separation and analysis of glycan species.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Depicts separation of 2-AB labeled glycans obtained from anti-VEGF antibody and spiked with system suitability standards
Figure 2. Depicts separation of commercially 2-AB labeled glycans
Figure 3. Depicts separation of 2-AB labeled glycans obtained from anti-VEGF antibody without system suitability standards
Figure 4. Depicts separation of 2-AB labeled glycans obtained from anti-HER2 antibody
Figure 5. Depicts separation of the Rapifluor labeled glycan species obtained from anti-VEFG and separated using the invented method
Figure 6. Depicts separation of the Rapifluor labeled glycan species from anti-VEFG and separated using the method already existing in the prior art.

LIST OF TABLES
Table 1. Depicts the saccharide composition and the alternate nomenclatures of the various glycan species
Table 2. Describes the disclosed gradient program used for separation of the glycan species
Table 3. Describes the gradient program present in the prior art
Table 4. Describes the detailed masses of various rapifluor labeled glycan species separated by the disclosed method and the prior art method
SUMMARY OF THE INVENTION
The present invention discloses a rapid method to separate various labeled species of glycans in a sample comprising monoclonal antibody (mAb) by hydrophilic interaction liquid chromatography (HILIC) using a salt gradient on a UPLC system. The said optimized method for the elution of species is performed by alteration of the flow rate based on the nature of the glycan species in the sample in a time dependent manner during the course of chromatography. The disclosed method provides an elution time of less than 35 minutes and higher resolution of various glycan species.
DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Various embodiments of the disclosed invention provide a rapid and sensitive UPLC based method to separate labeled glycan species in a sample.
In an embodiment the claimed invention describes a rapid and sensitive chromatographic method for separating various labeled glycan species such as, but not limited to those listed in the Table 1, wherein, the method involves the use of a salt gradient and series of varying flow rate on a hydrophilic column.
In one embodiment the invention discloses a rapid, UPLC based method for separation of labeled glycan species comprising,
a. Pretreatment of sample wherein the sample is digested to release the glycan species
b. Labelling of the released glycans in step (a) with a chromophore
c. Performing separation
i. using a salt gradient obtained by mixing acetonitrile and ammonium formate for elution and
ii. using a series of flow rate comprising 0.5 ml/min from 0 to 17 minutes, 0.3 ml/min. from 17 to 26 minutes, 0.35 ml/ min from 26 to 28 minutes, 0.45 ml/min from 28 to 29 minutes and 0.5 ml/min from 29 to 32 minutes on a hydrophilic column
In another embodiment the pH of the salt gradient is acidic
In yet another embodiment the acidic pH of the salt gradient is between 3.5 and 5.5
In yet another embodiment the percentage of ammonium formate increases at the rate of 0.2% to 1% per minute.
In one embodiment the sample is a glycoprotein, wherein the glycoprotein is derived from one or more protein-producing higher eukaryotic cell clones, and wherein the clone optionally produces an antibody.
In another embodiment, the glycoprotein is derived from human sera from one or more patients or subjects.
In another embodiment, the sample of the present invention is subjected to pretreatment which involves the release of the glycans from the sample by enzymatic or chemical treatment. Chemical pre-treatment involves hydrazinolysis or by alkaline ß-elimination,or via commercially available kit such as GlycoProfile™ IV Chemical Deglycosylation Kit.
Enzymatic pretreatment involves digestion with enzymes such as PNGaseF (Peptide N-glycosidase F), or by using Endoglycosidase H (or an enzyme with similar enzymatic activity, such as IgGZERO™) or Endoglycosidase F2, which cleave between the two N-Acetylglucosamines of the glycan core leaving the first monosaccharide attached to the protein.
In a further embodiment, the glycoprotein sample of the present invention is enzymatically treated with the PNGaseF enzyme commercially available as “Prozyme®”.
In another embodiment, the pretreated sample with glycan species is further labelled with chromophore to be detected by optical spectroscopic techniques such Spectrofluorimetric. The glycans are derivatized prior to chromatographic separation at the reducing termini by reacting with a dye that imparts optical property and makes them detectable. This technique known as glycan labeling involves dyes selected from 2-amino benzamide (2-AB), anthraanilic acid, pyrilium or the like.
In yet another embodiment the glycan species of present invention, is labeled using 2-AB labeling from commercially available Prozyme® labelling kit.
In another embodiment the claimed invention describes a rapid and sensitive chromatographic method for separating different 2-AB labeled glycan species wherein the method involves a salt gradient and alteration of flow rate.
In one embodiment, the glycan separation is performed using an optimal flow rate such in accordance with the per van Deemter curve. Accordingly the optimum flow rate of the present invention is that at which the Height Equivalent to a Theoretical Plate (HETP) is minimum and as the separation efficiency of a column is inversely proportional to the HETP, the minima of the van Deemter curve is the region where better separation of the sample is obtained
In one embodiment the invention discloses a rapid, UPLC based method for separation of 2-AB labeled glycan species comprising,
a. Pretreatment of sample wherein the sample is digested to release the glycan species
b. Labelling of the released glycans in step (a) with a chromophore
c. Performing separation
i. using a salt gradient obtained by mixing 100% acetonitrile and 100 mM formate for elution with acidic pH and
ii. Using a series of flow rate at 0.5 ml/min from 0 to 17 minutes, 0.3 ml/min. from 17 to 26 minutes, 0.35 ml/ min from 26 to 28 minutes, 0.45 ml/min from 28 to 29 minutes and 0.5 ml/min from 29 to 32 minutes.
The method as disclosed in the present invention helps perform glycan analysis of a sample by rapidly resolving and quantifying the labeled glycan species and reduces the analysis time per sample as compared to the existing HPLC based protocol.
In one embodiment, the optimized UPLC based method for separation of glycan species in a sample, provides effective resolution of glycans in the sample leading to lesser unidentified peaks and in turn effective characterization of glycan composition of said sample.
In an embodiment, the invention discloses a rapid and sensitive UPLC based method to separate, identify and characterize various labeled glycan species where in the method comprises
a. Labeling of the glycan species with a fluorophore
b. Separation of the labeled glycan species on a UPLC system using a salt gradient obtained by mixing 100% acetonitrile and 100 mM formate for elution with acidic pH and series of flow rate at 0.5 ml/min from 0 to 17 minutes, 0.3 ml/min. from 17 to 26 minutes, 0.35 ml/ min from 26 to 28 minutes, 0.45 ml/min from 28 to 29 minutes and 0.5 ml/min from 29 to 32 minutes on a hydrophilic column.
c. Characterization of the separated labeled glycan species
In one embodiment the characterization of the separated labeled glycan species is performed using various techniques known in the art including biophysical techniques such as mass spectrometry.
In another embodiment the labeled glycan species that is separated is selected from the species as listed in the table 1. The table details on the existing nomenclature and the nomenclature adopted to characterize glycans in the present invention

Glycan annotation
Glycan Species Saccharides Comp. mAb style Oxford
M3F (GlcNAc)2(Man)3(Fuc)1 M3F FA0
M3+NAG (GlcNAc)3(Man)3 Man3 (+B) A1
M3F+NAG (GlcNAc)3(Man)3(Fuc)1 G0F (-B) FA1
G0 (GlcNAc)4(Man)3 G0 A2
G0F (GlcNAc)4(Man)3(Fuc)1 G0F FA2
G1F-NAG (GlcNAc)3(Man)3(Gal)1(Fuc)1 G1F (-B) FA1G1
M5 (GlcNAc)2(Man)5 Man5 M5
G1A (GlcNAc)4(Man)3(Gal)1 G1 A2G1
G1B* (GlcNAc)4(Man)3(Gal)1 G1 A2G1
G0F+NAG (GlcNAc)5(Man)3(Fuc)1 G0FB FA2B
G1F (G1AF) (GlcNAc)4(Man)3(Gal)1(Fuc)1 G1F FA2G1
G1F (G1BF)* (GlcNAc)4(Man)3(Gal)1(Fuc)1 G1F FA2G1
M5F+NAG (GlcNAc)3(Man)5(Fuc)1 Man5F (+B) M5FB
G1F+NAG (GlcNAc)5(Man)3(Gal)1(Fuc)1 G1FB FA2G1B
M6 (GlcNAc)2(Man)6 Man6 M6
G1F+NAG* (GlcNAc)5(Man)3(Gal)1(Fuc)1 G1FB FA2G1B
G2 (GlcNAc)4(Man)3(Gal)2 G2 A2G2
G1FS1-NAG (GlcNAc)3(Man)3(Gal)1(Fuc)1(NeuAc)1 G1FS1 (-B) FA1G1S
G2F (GlcNAc)4(Man)3(Gal)2(Fuc)1 G2F FA2G2
M6+NAG (GlcNAc)3(Man)6 Man6 (+B) M6B
G1FS1 (GlcNAc)4(Man)3(Gal)1(Fuc)1(NeuAc)1 G1FS1 FA2G1S
G1FS1* (GlcNAc)4(Man)3(Gal)1(Fuc)1(NeuAc)1 G1FS1 FA2G1S
G2F+NAG (GlcNAc)5(Man)3(Gal)2(Fuc)1 G2FB FA2G2B
M6F+NAG (GlcNAc)3(Man)6(Fuc)1 Man6F (+B) M6FB
G2FS1-NAG (GlcNAc)3(Man)3(Gal)2(Fuc)1(NeuAc)1 G2FS1 (-B) FA1G2S
M7 (GlcNAc)2(Man)7 Man7 M7
G2FS1 (GlcNAc)4(Man)3(Gal)2(Fuc)1(NeuAc)1 G2FS1 FA2G2S
G2FS1* (GlcNAc)4(Man)3(Gal)2(Fuc)1(NeuAc)1 G2FS1 FA2G2S
M5F+NAG+G+S (GlcNAc)3(Man)5(Gal)1(Fuc)1(NeuAc)1 - M5FAG1S
M8 (GlcNAc)2(Man)8 Man8 M8
M8* (GlcNAc)2(Man)8 Man8 M8
G2FS2 (GlcNAc)4(Man)3(Gal)2(Fuc)1(NeuAc)2 G2FS2 FA2G2S2
M9 (GlcNAc)2(Man)9 Man9 M9
M3 (GlcNAc)2(Man)3 M3 A0
G2S (GlcNAc)4(Man)3(Gal)2(NeuAc)1 G2S A2G2S
G2S2 (GlcNAc)4(Man)3(Gal)2(NeuAc)2 G2S2 A2G2S2
Table 1
*: Positional isomers

Examples
The gradient program, as disclosed in the present invention for the separation of the glycans was developed by mixing said two solvents - acetonitrile (ACN) and 100 mM ammonium formate in varying compositions as provided in the Table 1 below. The mobile phase so obtained is allowed to be passed through the column at varying flow rates as provided in the Table 2.
Time (min.) Flow rate (ml/min) % of ACN % of 100mM ammonium formate
0 0.5 72 28
0.3 0.5 72 28
17 0.3 67 33
26 0.35 62 38
28 0.45 60 40
29 0.5 0 100
30 0.5 0 100
30.1 0.5 72 28
32 0.5 72 28

Table 2. The gradient program for the separation of Glycans on UPLC

.
Time (min.) Flow rate (ml/min) % of ACN % of 100mM ammonium formate
0 0.4 75 25
35 0.4 54 46
36.5 0.2 0 100
39.5 0.2 0 100
43.1 0.2 75 25
47.6 0.4 75 25
55 0.4 75 25

Table 3. The gradient program already existing in the art for the separation of Glycans on UPLC.
Example 1. Glycan preparation, labelling & UPLC based separation
Sample comprising antibodies were characterized based on the separation of various glycan species.
The sample comprising anti-VEGF antibody were treated with glycanase enzyme PNGase F and incubated at 37°C for 16- 18 hours. After incubation the glycans were purified using ethanol precipitation and labeled using commercially available Prozyme’s® 2-AB labeling kit and incubating at 65°C for 2.5 hours. The 2-AB labeled glycans were purified using S-cartridge cleaning kit. The released glycans were spiked with system suitability standards and reconstituted using 70% acetonitrile and analyzed on FLR UPLC using Waters BEH glycan column (1.7 µ-, 2.lx150 mm) on Waters UPLC according to the gradient program provided in Table 2. The chromatogram obtained is depicted in the Figure 1.
Additionally, commercially available 2-AB labeled glycan standards were separated by following the same protocol as a control. The chromatogram obtained is depicted in the Figure 2

Example 2. Glycan preparation, labeling & UPLC based separation of Anti-VEGF antibody.
The glycan obtained from Anti-VEGF antibody by protocol as provided in Example 1 were separated without the system suitability standards. The chromatogram so obtained is as depicted in the Figure 3.
Example 3. Glycan preparation, labeling & UPLC based separation of Anti-HER2 antibody.
Anti-Her2 antibody sample were subjected to the glycan preparation step and the glycans so obtained were subsequently separated as provided in Example 1. The chromatogram so obtained is as depicted in the Figure 4
Example 4. Comparison of the method disclosed in the invention with the method existing in the art.
The glycan samples were obtained from anti-VEGF antibody and labeled using the commercially available RapiFluor-MS N-Glycan Kit.
The anti-VEFG antibody samples were denatured with Rapigest detergent at 80º to 90º C for 3 minutes and then subjected to PNGase F digestion for deglycosylation at 50º C for 5 minutes. The glycans so obtained were labeled with fluorescent dye such as RF at room temperature for 5 minutes. The labeled glycans were cleaned up and eluted in 200 mM Ammonium acetate and 5% acetonitrile (ACN). The labeled glycans were separated and characterized on QDa detector in sequence with FLR UPLC equipped with Waters BEH glycan column (1.7 µ-, 2.l x 150 mm) using gradient program provided in Table 2. The chromatogram so obtained is as depicted in the Figure 5. Each species was identified using their mass.
The labeled glycan species as obtained in the above step were also separated and characterized on QDa detector in sequence with FLR UPLC equipped with Waters BEH glycan column (1.7 µ -, 2.lx150 mm) using gradient program already known in the literature as provided in Table 3. The chromatogram so obtained is as depicted in the Figure 6. Each species was identified using their mass. The masses of each glycan species obtained upon separation using the two methods described in the example 4 are as given in the Table 4.
Peak number Glycan species Theoretical mass Claimed (32 mins.) Method Existing (55 mins.) prior art Method
m/z, 2+, H+H 1.00002 BZ RT 1.00001 BZ RT
RFMS BZ m/z BZ m/z
1 M3F 684.77 684.79 6.6 684.79 10.6
2 M3+NAG 713.28 713.3 7.3 713.3 11.3
3 M3F+NAG 786.31 786.33 8.7 786.33 12.5
4 G0 814.82 814.84 9.5 814.84 13.1
5 G0F 887.85 887.87 10.9 887.87 14.2
6 G1F-NAG 867.34 867.36 12.3 867.36 15
7 M5 773.8 773.81 12.9 773.81 15.4
8 G1A 895.85 895.86 13.3 895.87 15.6
9 G1B 895.85 895.85 13.9 895.87 16
10 G0F+NAG 989.39 989.4 14.6 989.41 16.3
11 G1F (G1AF) 968.88 968.89 15.1 968.89 16.6
12 G1F (G1BF) 968.88 968.89 15.9 968.89 16.9
13 M5F+NAG 948.36 948.38 16.7 948.38 16.9
14 G1F+NAG 1070.42 1070.43 17.6 1073.43 17.7
15 M6 854.82 854.84 18 854.84 17.9
16 G1F+NAG 1070.42 1070.43 18.3 1073.43 18.1
17 G2 976.88 976.87 18.8 976.88 18.3
18 G1FS1-NAG 1012.89 1012.9 19.4 1012.9 18.4
19 G2F 1049.9 1049.92 20.7 1049.92 19.2
20 M6+NAG 956.36 956.38 21.4 956.38 19.5
21 G1FS1 1114.43 1114.44 21.8 1114.44 19.6
22 G1FS1 1114.43 1114.44 22 ND ND
23 G2F+NAG 1151.44 1151.47 22.52 1151.45 20.7
24 M6F+NAG 1029.39 1029.4 22.9 1029.41 20.4
25 G2FS1-NAG 1093.91 1093.93 23.4 1093.93 20.5
26 M7 935.85 935.86 23.6 935.82 14.2
27 G2FS1 1195.45 1195.47 24.8 1195.47 21.4
28 G2FS1 1195.45 1195.47 25 1195.47 21.6
29 M5F+NAG+G+S 1174.957 1174.94 25.98 1174.94 22.2
30 M8 1016.88 1016.89 26.6 1016.9 22.8
31 M8 1016.88 1016.89 27 1016.6 23.1
32 G2FS2 1341 1341.01 27.8 1341.02 23.6
33 M9 1097.9 1097.91 28.8 1097.92 24.9
Table 4
,CLAIMS:We Claim:
1. An Ultra Performance Liquid Chromatography based method for separation of glycan species from a sample comprising,
a. Pre-treatment of sample wherein the sample is digested to release the glycan species
b. Labelling of the released glycans in step (a) with a chromophore
c. Performing separation
i. using a salt gradient obtained by mixing acetonitrile and ammonium formate for elution and
ii. using a series of varying flow rates comprising 0.5 ml/min from 0 to 17 minutes, 0.3 ml/min. from 17 to 26 minutes, 0.35 ml/ min from 26 to 28 minutes, 0.45 ml/min from 28 to 29 minutes and 0.5 ml/min from 29 to 32 minutes on a hydrophilic column
wherein the series of varying flow rates used provide effective resolution of glycans in the sample.
2. A method according to claim 1, wherein the pH of said salt gradient is acidic.
3. A method according to claim 1, wherein the pH of said salt gradient is between 3.5 and 5.5.
4. A method according to claim 1, wherein the said sample is glycoprotein.
5. A method according to claim 4, wherein the said glycoprotein in an antibody.
6. A method according to claim 5, wherein the said antibody is anti-VEGF or anti-HER2.
7. A method according to claim 1, wherein the said chromophore is 2-Aminobenzamide or RapiFluor-MS.
8. A method according to claim 1, wherein the total run time is about 32 minutes.
9. An Ultra Performance Liquid Chromatography based method for separation of glycan species from a sample comprising,
a. Pre-treatment of sample wherein the sample is digested to release the glycan species
b. Labelling of the released glycans in step (a) with a 2-Aminobenzamide
c. Performing separation
i. using a salt gradient obtained by mixing 100% acetonitrile and 100 mM formate for elution with acidic pH and
ii. using a series of varying flow rates at 0.5 ml/min from 0 to 17 minutes, 0.3 ml/min. from 17 to 26 minutes, 0.35 ml/ min from 26 to 28 minutes, 0.45 ml/min from 28 to 29 minutes and 0.5 ml/min from 29 to 32 minutes.
wherein the series of varying flow rates used provide effective resolution of glycans in the sample.
10. An Ultra Performance Liquid Chromatography based method for separation of glycan species from a sample comprising,
a. Pretreatment of sample wherein the sample is digested to release the glycan species
b. Labeling of the glycan species with RapiFluor-MS
Separation of the labeled glycan species on a UPLC system using a salt gradient obtained by mixing 100% acetonitrile and 100 mM formate for elution with acidic pH and series of varying flow rates at 0.5 ml/min from 0 to 17 minutes, 0.3 ml/min. from 17 to 26 minutes, 0.35 ml/ min from 26 to 28 minutes, 0.45 ml/min from 28 to 29 minutes and 0.5 ml/min from 29 to 32 minutes on a hydrophilic column,
wherein the series of varying flow rates used provide effective resolution of glycans in the sample.
d. Characterization of the separated labeled glycan species